The method relates to a method for the analysis of macromolecules using a microarray.
Such a method for the rapid analysis of macromolecules comprises the application in a regular arrangement on a so-called microarray. The densities which can presently be achieved on such a microarray are, for example, for DNA (deoxyribonucleic acid) up to 250,000 different species of molecules per cm2. A parallel and rapid deposition of molecules can be achieved, for example, using so-called pipetting robots. An analysis, e.g. a fluorescence measurement, can easily be made in such an array geometry (“DNA Microarray”, published by M. Schena, Oxford University Press, New York, 1999). Substances studied are, for example, antibodies, antigens, proteins or DNA.
In such a microarray different species of macromolecules are arranged at different locations in a matrix form. A liquid with another species of macromolecules is washed over the microarray and forms a specific bond with at least one species of macromolecules on the microarray. If the liquid is removed from the surface again, the macromolecules to be studied are only retained at the locations of the specific binding. With the aid of a spatially resolved measurement, e.g. a fluorescence measurement, it is possible to determine the locations at which the macromolecule to be studied is present. From the known position of the individual macromolecules in the matrix form of the microarray it is also possible to determine the species of macromolecules with which the macromolecule to be studied has formed a specific bond.
In this case, it must be ensured that the macromolecules to be studied can come in contact with all macromolecules in the matrix form. For this purpose, for example, the entire surface on which the microarray is located must be flooded with the liquid containing the macromolecule to be studied.
The duration of a corresponding experiment is determined by the diffusion and can thus take some time. If, for example, the concentration of the macromolecule to be studied in the liquid is only low, it can take a very long time before it has found its specific binding partner on the array. In addition, the reaction kinetics of the specific binding depends on many reaction parameters, such as for example, the temperature, the salt concentration and/or the pH which are difficult to keep constant over a fairly long time on an area of typically a few 100 μm2 to a few cm2. In order to improve the resulting limited reproducibility of the results, presently a plurality of the same molecular species are frequently provided at different locations on the microarray so that statistical predictions can thus be made.
The liquid with the macromolecule to be studied, e.g. an oligonucleotide can also be sent over a solid surface through microchannels along which macromolecules of different species are located as capture oligonucleotides (“Geniom® Technology: From DNA Chips to DNA Processors” in “Chips to Hits”, 06-09 Nov. 2000, Philadelphia, Pa., USA; IBC Conferences Inc.).
The oligonucleotide to be studied thereby comes into the vicinity of all the capture oligonucleotides along the channel.
For such a prior art the microarray is, for example, a sandwich structure with external pumps on whose underside is located a CCD camera. The bottom and lid are transparent so that the fluorescence of the labelled sample oligonucleotide can be measured in the transmitted light. The liquids with the various oligonucleotides are pumped through closed channels on the array. In view of the fact that a typical channel cross-section is 100 μm by 100 μm, a pressure of several bar is required for the necessary lengths of the channels, as a result of which both the feeds and the chip itself are expensive. The pumps and valves which carry this out, necessarily have a dead volume which leads to an increased consumption of reagents.